Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection

Milk proteins determine important milk technological characteristics. Among caseins, Ƙ-casein has been correlated with fat and protein content and cheese yield. Fourteen Ƙ-caseins variants have been described but the alleles A, B and E are the most important ones due to their frequency and/or influence on the technological aptitudes of milk. Therefore, in the present study two different duplex qPCR assays with locked nucleic acid probes (for positions 13104 and 13124 of the Ƙ-casein gene) were developed for the detection of A, B and E variants. Firstly, DNA isolation method from milk somatic cells and hair was optimised. The developed 13124-qPCR assay showed an increased sensitivity reaching up to 6.7 copies DNA copies/reaction at a 95% confidence level with A, B and E alleles reference samples. The 13104-qPCR assay reached up to 6.7 DNA copies/reaction for A allele reference sample and 67 DNA copies/reaction for B and E samples. Intra-assay variation results were below 6%. Applicability was determined using DNA samples from animals with known genotype for Ƙ-casein (AA, AB, BB, BE, AE, EE) and both assays were able to discriminate among the six genotypes with 100% accuracy. Thus, this qPCR method represents a sensitive and rapid option for the detection of Ƙ-casein alleles in both hair and milk samples.

. Duplex real-time PCR standard curves using the A synthetic reference DNA sample (blue colour) and the AA homozygous genotyped sample (green colour). The standard curves were made by plotting Cq values against log10 DNA copies/reaction of each type of sample. Each data point represents the averaged Cq value of three replicates. Table S1.
Intra-and inter-assay repeatability of duplex real-time PCR assay for A, B and E allele discrimination of the CSN3 gene using synthetic reference DNA samples of A, B and E alleles and 13124 and 13104 probes.

Milk samples pre-treatment and pre-lysis step
Fresh milk samples were pre-treated based on the method described by Yap et al. (2020). Briefly, 2x10 ml milk samples were centrifuged at 4500 g for 20 min at 4°C, the fat was removed by a sterile spatula and the supernatant was discarded. The two somatic cell pellets were pooled and resuspended in 800 µl phosphate-buffered saline (PBS, pH 7.4), before centrifugation at 4500 g for 1 min at room temperature. Subsequently, the supernatant was discarded and the pellet was washed twice in 1 ml of PBS. The somatic cells pellet was then stored at 20°C for 20 min. Pre-lysis step of unfreeze pellets was carried out according to NucleoSpin Tissue kit instructions. Unfreeze pellets were resuspended in 180 μL of T1 buffer, and then, 25 μl of Proteinase K were added, vortexed and incubated at 56°C for 2 hours. After pre-lysis, the DNA extraction continued with the lysis step described below.

Hair samples pre-lysis step
Hair roots from each individual sample was put into a 1.5 ml microcentrifuge tube. After that, two prelysis steps were carried out according to the NucleoSpin Tissue manufacturer's instructions. Firstly, 360 µL of Buffer T1 were added, then, a rapid freezing step with nitrogen liquid followed by a subsequent thawing in a thermoblock at 56 ºC were performed four different times. In the second pre-lysis step, 25 μl of Proteinase K were added, vortex mixing was performed and the samples were incubated at 56°C overnight. After pre-lysis, the DNA extraction continued with the lysis step described below. DNA extraction from milk somatic cells and from hair samples was performed using the commercial NucleoSpin Tissue kit (Machenerey-Nagel) (Psifidi et al., 2010), following the manufacturer's instructions. After incubation, for lysis step, 200 μL of lysis Buffer B3 were added to the samples and they were incubated at 70°C for 10 min. Subsequently, to adjust DNA binding conditions, 210 μl of cold ethanol (96-100%) was added. Samples were then added to the NucleoSpin Tissue columns kit and centrifuged 1 min at 11000 g. The eluate was discarded. The aim of this last step was to remove the remaining components that were not DNA. Furthermore, this DNA purification was repeat, but this time, by adding 500 μl of Buffer BW. A second wash of the silica membrane was performed with 600 μL of Buffer B5 with ethanol and centrifuged again at 11000 g for 1 min. This last wash was performed in duplicate. To dry the silica membrane and remove residual ethanol from the silica membrane, the column was centrifuged at 11000 g for 1 minute. Finally, once all residues were removed, the DNA was eluted from the silica membrane into a 1.5 mL microcentrifuge tube. For this, 50 µL of Elution Buffer BE (preheated to 70°C) was used, incubated at room temperature for 1 minute and centrifuged for 1 minute at 11000 g. Each 1.5 ml microcentrifuge tube should contain approximately 50 μL of extracted DNA.

Target Oligonucleotide
Sequence (

S2. DNA copy number calculation
Standard curves were created by plotting the Cq against the log10 of the DNA copy number 59 . The copy number for each synthetic reference DNA sample (A, B and E) were calculated according to the manufacturer instructions (IDT, Coralville, USA) with the following formula: = ( ) ( ) ( 1 10 −15 ) 6.022 10 23 where C is the concentration in ng/µL of the synthetic reference DNA samples (10 ng/µL), M is the molecular weight in fmol/ng (11.18 fmol/ng), and 6.022 x 10 23 is the Avogadro constant.
In the case of AA genotyped DNA copy number were calculated as described previously 40,59 : = ( ) 6.022 10 23 ( ) 10 9 where 6.022 x 10 23 is the Avogadro constant, bovine genome size was considered to be 3 billion base pairs and the average molecular mass per base pair was defined as 660 g/mol.